Organ Preservation with Histidine-Tryptophan-Ketoglutarate (HTK) Solution with an Emphasis on Non-Heart Beating Donors (NHBD)

1
Organ Preservation with Histidine-Tryptophan-Ketog
lutarate (HTK) Solution with an Emphasis on
Non-Heart Beating Donors (NHBD)

• John J. Fung, M.D., Ph.D.
• Cleveland Clinic Foundation
• Transplant Center

2
Organ Preservation

• Preservation solutions are used to maintain the
  organ in optimal condition from the time of
  explantation until transplantation

3
Ischemia

• Decreased mitochondrial function
• Anaerobic conditions - depletion of ATP
• Alterations in ion permeability
• Accumulation of lactate
• Accumulation of hypoxanthine
• Cell swelling
• Cytosolic calcium accumulation

4
Reperfusion

• Generation of reactive oxygen species
• Increased oxidative stress
• Lipid peroxidation of cellular membranes
• Free radical formation leads to cellular
  destruction
• Results in macrophage/Kupffer cell activation
• Increased serum tumor necrosis factor (TNF)
• Damage can lead to prolonged hypoxia after
  reperfusion

5
Principles of Liver Preservation

• Exsanguination to reduce intravascular thrombosis
• Hypothermia to reduce cellular metabolism
• Maintain cell membrane integrity to avoid
  cellular swelling
• Susceptibility to cold ischemic injury vascular
  endothelium gt hepatocytes

6
History of Organ Preservation

• Simple cooling with cold solution
• Continuous hypothermic perfusion
• Collins (1967)
• Euro-Collins (1980)
• University of Wisconsin - ViaSpan (1986)
• HTK - Custidiol (1980s)
• Celsior - 1994

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8
Euro-Collins Solution
High potassium, glucose, and phosphate-based
solution Designed to mimic composition of
intracellular fluid Low cost Poor preservation
quality Short preservation times achievable
9
UW Solution
Use of impermeant molecules, lactobionate and
raffinose, in preventing cell swelling First
developed for and applied in preservation of
canine pancreas Hydroxyethyl starch to minimize
interstitial edema during machine perfusion, not
necessary during cold storage High K, low
Na
10
Southard and Belzer
11
UW Solution Disadvantages
Glutathione is oxidized during storage
addition of fresh GSH immediately before use
other additives High viscosity Solution
cannot be released into circulation (high K
content) Huge particles 100 µm in diameter
contained in original solution must use in-line
filtration with 40 µm pore size.Particles caught
in capillary bed of perfused organ, resulting in
vascular constriction, impeded reperfusion, and
reduction of functional recovery
12
Crystals in UW solution stored at sub-zero
temperatures (a ) perfused livers (b)
pancreas (c) kidneys (d)
Tullius et al AJT 2627
13
M.M. Gebhard, H.J. Kirlum, C. Schlegel. Organ
preservation with the solution HTK
14
HTK Solution (Custodiol)

• Developed as cardioplegia
• Low potassium, high sodium
• No colloid - viscosity equal to that of pure
  water from 1 to 350C, with mean flow rate 3X that
  of UW solution at equal perfusion pressure -
  organs exsanguinate and cool down to lower
  temperatures more rapidly than with UW

15
Status of Clinical Use of HTK Solution
(Custodiol?)
Estimated Clinical Volume (by 2004) Principal Location of Clinical Use Studies prior to routine use
Cardioplegia Open heart surgery gt1,000.000 90 centers worldwide routinely used retrospective studies
Transplantation gt 20,000 Europe/Asia/US single center studies
Kidney protection In-situ surgery gt 100 Europe single case studies
Related transplantation gt 4,000 Europe/Asia/US single center studies
Unrelated transplantation gt6,000 Eurotransplant/ US multi-center pilot study single case reports
Liver protection gt 600 Germany single center studies
Ex-vivo-surgery gt 150 Germany single center studies
Pancreas transplantation gt 1,000 US/Europe single case reports
16
Kidney Transplantation

• de Boer et al Eurotransplant randomized
  multicenter kidney graft preservation study
  comparing HTK with UW and Euro-collins.
  Transplant Int, 1999, 12447
• UW (168) vs EC (155) vs HTK (336),
• DGF 33 UW vs 43 EC vs 31 HTK
• 3-year Graft survival UW (68), HTK (73),
  
• EC (67)
• Conclusions HTK is comparable to UW in its
  preservative capabilities in cadaveric renal
  transplantation
• Factors influencing DGF Donor age, cause
  of death, CIT

17
J. De Boer, et al. Eurotransplant randomized
multicenter kidney graft preservation study
comparing HTK, UW and Euro-Collins (Transplant
Int, 1999)
18
J. De Boer, et al. Eurotransplant randomized
multicenter kidney graft preservation study
comparing HTK, UW and Euro-Collins (Transplant
Int, 1999)
19
J. De Boer, et al. Eurotransplant randomized
multicenter kidney graft preservation study
comparing HTK, UW and Euro-Collins (Transplant
Int, 1999)
20
J. De Boer, et al. Eurotransplant randomized
multicenter kidney graft preservation study
comparing HTK, UW and Euro-Collins (Transplant
Int, 1999)
21
Liver Transplantation

• Hatano et al Hepatic preservation with
  histidine-tryptophan-ketoglutarate solution in
  living-related and cadaveric liver
  transplantation. Clinical Science (1997), 9381
• LRD liver HTK (15) vs UW (49)
• CAD liver HTK (30) vs UW (18)
• Lower transaminases
• Improved bile flow
• No difference in survival or rejection rates

22
E. Hatano, et al. Tissue oxygenation in living
related liver transplantation (Clinical Science,
1997)
LRLT
Intraoperative changes in mean value of oxygen
saturation of Hb at 10 points in liver graft
After reflow
of operation
23
E. Hatano, et al. Tissue oxygenation in living
related liver transplantation (Clinical Science,
1997)
Cadaveric
Intraoperative changes in mean value of hepatic
tissue oxygen saturation of Hb at 10 points in
liver graft
24
E. Hatano, et al. Tissue oxygenation in living
related liver transplantation (Clinical Science,
1997)
LRLT
CV of oxygen saturation of Hb at 10 points in
liver graft, indicating heterogeneity of tissue
oxygenation
After reflow
of operation
25
E. Hatano, et al. Tissue oxygenation in living
related liver transplantation (Clinical Science,
1997)
26
Liver Transplant Patient Survival Hannover (1988
- 2000)

HTK (n 400)
UW (n 4 92)
P lt 0.0331 (LogRank)
years
27
Liver Transplants Graft Survival Hannover (1988
- 2000)

HTK (n 461)
UW (n 607)
P lt 0.0029 (LogRank)
years
28
HTK and UW for Liver PreservationHannover (1988
- 2000) n 1068

• lt 5 days after transplantation HTK
  UW
• n 461 607
• PF 439 578
• INF 22 29
• INF 4.8 4.8
• p 1.00

29
HTK and UW for Liver PreservationHannover (1988
- 1998) n 836

• Biliary Tract Complications HTK
  UW
• n 305 531
• BTC 39 65
• BTC 12.8 12.2

30
HTK and UW for Liver PreservationHannover (1988
- 2000) n 1068

• CIT gt15 hours HTK UW
• n 36 154
• PF 34 143
• INF 2 11
• INF 5.6 7.1
• p 1.000

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HTK vs. UWPatients and Methods
Patients 123 120 Adults, 3 Children
Age 1 - 70 years Transplantations Total
134 Cadaveric 123 primary, 10 secondary, 1
tertiary 114 standard orthotopic, 5 split, 4
partial Living donation 11 (right
lobe) Combined 6 kidney transplantation 1
bone marrow transplantation 1 heart and
kidney transplantation Preservation solution 63
HTK und 71 UW
32
HTK vs. UWInitial Liver Function
HTK UW OLT total 63
71 Initial function (IF) 45 (71.5) 43
(60.5) Initial dysfunction (IDF) 13
(20.6) 26 (36.6) Initial nonfunction (INF)
5 (7.9) 2 (2.8)

33
HTK vs. UWBiliary Complications
HTK UW Bile duct necrosis 3 (16, 17,
485 d) 3 (44, 10, 8, 46 d) Localized
strictures 2 (72, 150 d) 2 (210, 305
d) Diffuse strictures (ITBL) - 3 (610, 210,
365 d) Total 5 8 ITBL ischemic type
biliary lesion
34
HTK vs. UW Biochemical Parameters
HTK UW AST max (U/l) 1320 1254
1389 1214 pod 7 (U/l) 26.7
17.5 24.3 18.4 AP pod 7 (U/l) 159.7
94.6 214.8 109.2 GGT pod 7 (U/l)
81 52.9 84.6 59.5 Bilirubin pod 14
(mg/dL) 9.5 9.7 13.8 12.6

35
Pittsburgh Protocol

• Trial of HTK in 100 consecutive standard
  multiorgan donors - comparison group was historic
  (UW) age matched controls (21) for age, CIT,
  organs removed and transplanted using CORE donor
  database. Outcomes to include
• Patient and graft survival
• Initial function vs. DGF vs. PNF
• HTK was used on all NHBD and liver alone donors

36
Patients

• HTK
  UW
• Donors 84
  169
• Gender 49 M, 35 F
  98 M, 71 F
• Age 2 Wks - 75Y
  3Y - 75Y
• (47)
  (46)
• (43.3 19)
  (43.7 18.8)
• Recipients
• Gender 62 M, 22 F
  101 M, 68 F
• Age 2Y - 71Y
  1Y - 73Y
• (50)
  (52)
• (49.7 13)
  (50.3 12.3)

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Liver Transplantation, CadavericUW / HTK

• Age D/R 43.718.8 (3-75) / 50.312.3
  (1-73)
• Age D/R 43.319 (1wk-75) / 49.713 (2-71)
• CIT 619191
• CIT 616193
• AST1 12932907 Median (597)
  (97-31490)
• AST1 1489.22404
  (595) (72-15750)
• ALT1 834.91192
  (483) (91-6552)
• ALT1 914.81131.3
  (450) (85-7016)
• AST7 99.9189.2
  (51) (17-1969)
• AST7 72.964.4
  (55) (12-498)
• ALT7 189.4191.2
  (137) (40-1408)
• ALT7 178.9145.6
  (139) (22-568)

38
Graft Function

• HTK
  UW
• 84 ()
  169 ()
• IGF 66 (78.5)
  134 (79)
• DGF 16 (19)
  15 (9)
• PNF 2 (2.2)
  20 (11.8)
• CIT gt 14 hours 13 ()
  27 ()
• IGF 8 (62)
  19 (70)
• DGF 3 (23)
  5 (18.5)
• PNF 2 (15)
  3 (11)

39
30-day Survival

• HTK
  UW
• 84
  169
• Graft 94
  83
• Patient 96
  89
• Re-OLTX 3 13
• in 30 days
• HAT 2
  4

40
HTK vs. UW in LDLT

• Chan et. al. Liver Transplantation 2004
  101415-1421 (Hong Kong)
• UW
  HTK
• Number of patients 30
  30
• Age 38.5
  35.5
• CIT 112 (79
  334) 111.5 (75 222)
• Biliary stricture 10 (33)
  6 (20)
• Pre-reperfusion flush Yes
  No
• Graft loss 0
  1
• Hospital mortality 0
  0
• Biochemicals Same, except PT (higher in HTK)
• Cost analysis UW 137.6 higher than HTK/patient
• Not significant

41
HTK vs. UW in LDLTA Prospective Study

• Testa et. al. Liver Transplantation 2003
  9822-826 (Chicago and Essen)
• Donors Right lobe Age (33 10) 18 M, 12
  F
• Recipients Age (49 9), 20 M, 10 F, MELD
  13.4 7.4
• Mean Follow-up 13 7 months
  
• UW
  HTK
• Perfusion 14
  16
• Artery 1 mL/g
  3 mL/g
• Portal vein 1 mL/g
  1.5 mL/g
• Pre-reperfusion flush Yes
  No (practical advantage)
• CIT 147 44
  144 40
• Patient survival 79
  88
• Graft survival 72
  83
• PNF 1
  0
• HAT 1
  1
• Liver biochemical values Similar
• No intrahepatic biliary stricture
• Overall Equally effective. HTK has practical
  and economical advantage

42
HTK solution for organ preservation in human
liver transplantationA prospective multi-center
observation study

• Pokorny et. al. Transplant International 2004
  17256-260 (Austria, Germany)
• 214 patients in 4 European centers (1996-1999)
• 5 liters of HTK for preservation CIT 444 224
• All vascular anastomoses completed before
  reperfusion
• No pre-reperfusion flush
• PNF 2.3, Initial dysfunction 6.5
• Graft dysfunction not correlated with CIT
• 1-year patient and graft survival 83 and 80
  (unrelated to CIT)
• HTK safe and effective and easy to use.
• Comparable to UW with less cost.

43
HTK vs. UW in liver transplantationA meta
analysis
Feng et.al. Liver Transplant, 2007
44
HTK vs. UW in liver transplantation A meta
analysis
P 0.87 RR 1.01
Patient Survival
P 0.86 RR 1.01
Graft Survival
Feng et.al. Liver Transplant, 2007
45
HTK vs. UW in liver transplantationA meta
analysis
Feng et.al. Liver Transplant, 2007
46
HTK vs. UW in liver transplantationA meta
analysis

• Cost HTK cheaper than UW
• Biliary complications
• Trend for less biliary strictures with HTK
• PNF, PDF, DGF No difference
• Graft survival No difference
• Patient survival No difference
• Biochemical values No difference

Feng et.al. Liver Transplant, 2007
47
Kidney Transplantation, Cadaveric(HTK)

• Total of 198 kidneys recovered, 155 used
• Immediate function (53) CIT 22 Hrs
• Fair function (30.6)
• ATN (16.4) CIT 28
  Hrs
• All functioning
• One kidney thrombosed shortly after transplant
  (technical)
• One kidney lost (hyperacute rejection)
• 39 kidneys were sent out of the OPO area
• Kidneys from NHBD 75 primary function
• (12 transplanted, 4 discarded) 25
  ATN with late function

48
Pancreas Transplantation, Cadaveric(HTK)

• 40 pancreata recovered
• 20 used
• 20 research
• 19 used locally
• One sent to another center (out of OPO) with
  kidney for K-P transplant with good function

49
HTK vs. UW in Pancreas Transplantation

• Potdar et al. Clinical Transplantation
  200418661-665 (University of Pittsburgh)

50
Kidney Transplantation, Cadaveric(HTK)

• Total of 198 kidneys recovered, 155 used
• Immediate function (53) CIT 22 Hrs
• Fair function (30.6)
• ATN (16.4) CIT 28
  Hrs
• All functioning
• One kidney thrombosed shortly after transplant
  (technical)
• One kidney lost (hyperacute rejection)
• 39 kidneys were sent out of the OPO area
• Kidneys from NHBD 75 primary function
• (12 transplanted, 4 discarded) 25
  ATN with late function

51
Biliary Complications After Liver Transplantation
52
Long-term Biliary Complications(one year)

• HTK
  UW
• (76)
  (134)
• Anastomotic stricture 3
  5
• Diffuse (ischemic) stricture 0
  2
• Bile leak 1
  1

53
Post-liver Transplant Biliary Strictures

• Biliary strictures after liver transplantation
  10-30
• Adequate flushing of peri-biliary arterial tree
  is important
• High viscosity preservation solutions might not
  completely flush the small donor peri-biliary
  plexus

54
Blood Supply to Extrahepatic Biliary Tree
55
Peri-Biliary Vascular Plexus

• Alpini et al.

56
Methacrylate Injection Replica of Peri-Biliary
Capillary Plexus

• Ohtani

57
NHBD and Liver Transplantation

• Abt et al. (Ann. Surg. 2004)
• UNOS database 144 NHBD vs. 26856 HBD
• 1Y
  3Y
• Graft survival 70.2 (80.4)
  63.3 (72.1)
• Patient survival 79.7 (85)
  72.1 (77.4)
• Increased incidence of PNF 11.8 vs. 6.4
• Increased re-transplantation 13.9 vs. 8.3
• Prolonged CIT predictor of early graft
  dysfunction
• More than 8 hours 30.4 graft failure (lt60
  days)
• More than 12 hours 58.3 graft failure
• Less than 8 hours 10.8
• Donors older than 60 Increased graft failure
  (25)

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Super Rapid Technique

• Cannulation of aorta
• Perfusion with HTK
• Topical cooling
• Thoracotomy
• aortic clamping
• drainage IVC
• En bloc removal and benchtable look for aberrant
  vessels

A. Casavilla, Pittsburgh, 1995
59
Liver Transplantation from NHBD

• 1993-1996
• 21 donors 17 recovered 9 used
  (55)
• Patient survival 4/9 (45)
• 1997-2001
• 43 donors 37 recovered 23 used
  (62)
• Patient survival 19/22 (86.5)
• 1/02-12/03
• 25 transplants (12 sent livers, 13 local)
• CIT sent vs. local 766 (min) 590
  (min)
• 2 DGF and one PNF (sent), one DGF (local)
• Patient survival 20/25 (80)

60
Liver Transplantation from NHBDDonor Procedure

• Time off the ventilator to pronounced
• 8-53 min. (2211)
  Median 19
• Time pronounced to cross-clamp
• 1-13 min. (65)
  Median 5
• Three patients had CPR for low blood pressure in
  the OR before withdrawal of support
• One patient with CPR for cardiac arrest at the
  time of prep. in the OR
• Flushes with UW solution 3000-5000cc
• HTK solution 6000-9000cc

61
Liver Transplantation from NHBDDonor Outcome

• Donors older than 60
• Four livers transplanted
• One PNF, sepsis (died)
• One HAT and biliary strictures (alive with
  problems)
• One delayed graft function, prolonged ICU stay,
  HD (alive)
• One normal function
• More than 10 discarded for different reasons
• Donor weight more than 85 Kg
• 8/13 discarded (62), mostly steatosis

62
Liver Transplantation from NHBDRecipient
Characteristics

• Fifty-seven transplants in 56 recipients
• 32 males 24 females (One patient received 2
  livers from NHBD)
• Age 18-69 y (5011) Median 50
• Primary disease HCV, PBC, AI, ETOH, ..
• CIT (min) 630144 (358-1056) Median
  600

63
Liver Transplantation from NHBD

• Post-transplant labs.
• Range MeanSD
  Median
• AST(peak) 77-13692 36203973
  2039
• ALT(peak) 96-7245 13551825
  504
• AST(day7) 14-285 6773
  41
• ALT(day7) 28-191 9345
  90
• PT(day7) 11-15
  13.11.1 12.8
• T.Bili. D/C 0.3-1.7
  0.90.4 0.95

64
Liver Transplantation from NHBDComplications

• Primary non-function (5) (9)
• 2 Re-OLTX (alive)
• 1 Re-OLTX (died)
• 2 died of sepsis
• Biliary complications (11) (19)
• Intrahepatic strictures, bile cast 6 (10)
• 2 Re-OLTX and alive
• 2 re-listed, one alive , one died
• 2 required percutaneous dilatation, both alive
• Anastomotic biliary reconstruction (5) All
  alive

65
Liver Transplantation from NHBDComplications

• Hepatic artery complications (8) (14)
• Hepatic artery stricture (2) Repaired
  Both alive
• Hepatic artery thrombosis (6) (11)
• Re-OLTX (3)
  Two died, one alive
• Repaired (1)
  Alive
• None (sepsis) (2)
  Died

66
Liver Transplantation from NHBDSurvivalPittsburg
h Data

• 1-Y
  5-Y 10-Y
• Patient survival 79 76
  76
• HBD (86)
  (72) (60)
• Graft Survival 68 65
  65
• HBD (78) (70)
  (56)

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Non-Heart-Beating Donors

• HTK
  UW
• 8
  15
• IGF 8
  12
• PNF 0
  3
• Biliary complications 1
  3
• Anastomotic 1
  1
• Diffuse 0
  1
• Bile leak 0
  1

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HTK (NHBD) UW
69
DCD Donor Liver Transplants1995 - 2004
184
Year of Transplant
70
Kaplan-Meier Adult Graft Survival Primary
Deceased Donor Liver Transplants 1998-2003
N22,199
N303
Includes adult, primary, liver alone transplants
71
Kaplan-Meier Adult Graft Survival Primary
Deceased Donor Kidney Transplants 1998-2003
N32,888
N1,177
N6,610
Includes adult, primary, kidney alone transplants
72
Yamauchi et al Warm preflush with streptokinase
improves microvascular procurement and tissue
integrity in liver graft retrieval from
non-heart-beating donors.Transplantation. 2000,
691780

• After 30 min of warm ischemia, microvascular
  perfusion of UW solution was found markedly
  altered when compared with that of sham-operated
  living controls
• Preflush with RL (RLUW) only slightly attenuated
  the perfusion disorder
• the addition of SK (7500 IU/100 cc) to RL
  (SK/RLUW) resulted in a significant improvement
  of microvascular graft perfusion (Plt0.05).
• the increased enzyme release observed in solely
  UW-flushed livers after 24 hr cold preseravtion
  was only slightly influenced by preflush with RL,
  but markedly attenuated (Plt0.05) by pre-flush
  with RL containing SK

73
Gok et al How to improve the quality of kidneys
from non-heart-beating donors a randomised
controlled trial of thrombolysis in
non-heart-beating donors.Transplantation. 2003
Dec 2776(12)1714-9

• 2 groups of NHBD donors -streptokinase and
  placebo
• 72 Maastricht II (uncontrolled), 28 Maastricht
  III (controlled)
• Streptokinase given at 1,500,000 units prior to
  preservation solution
• streptokinase-treated kidneys had a better
  appearance at procurement (Plt0.001)
• performed better during machine preservation
  (Plt0.001).
• higher proportion of kidneys transplanted through
  the use of streptokinase (63.6 with
  streptokinase vs. 42.6 with placebo)
• PNF - 0 streptokinase vs 17 placebo
• Graft function at one year - 93 streptokinase vs
  83 placebo

74

• Abdominal Organ Recovery (Adult Donor)
• Cannulate aorta, flush abdominal organs with 3-4
  liters of ViaSpan
• Cannulate portal, flush with 1-2 liters of
  ViaSpan
• PRESERVATION TIME 12-15 MINUTES
• Flush 1 additional liter of ViaSpan through liver
  during preparation for transportation
• Store liver in 1 liter of ViaSpan
• Kidneys stored in 1 liter of EuroCollins
• Solution Used 6-8 liters of ViaSpan
• 1 liter of Euro-Collins

75

• Abdominal Organ Recovery (Adult Donor)
• Cannulate aorta, flush abdominal organs with 8-10
  liters of HTK
• No Portal Canulation
• PRESERVATION TIME 4-7 minutes
• Flush 1 additional liter of HTK through liver
  during preparation for transportation, also used
  as storage solution
• Use 1 liter of HTK for storage of Kidneys
• Solution Used 10-12 liters of Custodiol HTK

76
COST COMPARISONViaSpan/Eurocollins vs. Custodiol
HTK

• ViaSpan with additives 327.00 Liter
• EuroCollins with additives 37.50 Liter
• PER CASE COST
• 6-8 Liters of ViaSpan 1962.00-2616.00
• 1 Liter of EuroCollins 37.50
• TOTAL 1963.50-2653.50
• Custodiol HTK 150.00 Liter
• PER CASE COST
• 8-10 Liters of Custodiol HTK 1200-1500
• POTINTIAL SAVINGS 763.50-1,153.50 PER CASE

77
ANNUAL PROJECTED COST SAVINGS USING CUSTODIOL HTK

• 73,000-137,000
• Based on 130 Cases

78
HTK vs. UWCost analysis and savings

• Englesbe et. al. Transplantation 200682580-581
  University of Michigan
• 
  Gift of
  Life, Michigan
• Material costs of 77 consecutive kidney-pancreas
  recovered for transplantation
• Significant cost reduction 43 actual cost
  saving for Gift of Life Michigan

79
IDENTIFIED ADVANTAGES OF CUSTODIOL HTK

• No additives
• Ready to use immediately for unstable donors
• No discarding of solution if case is aborted
• Packaging size
• No apparent difference in organ function
• Cost Savings
• Transport/storage at room temperature
• Approved for liver, kidney, pancreas and heart
  preservation

80
Conclusions Comparison of HTK and UW

• Overall, logistical and cost benefits with HTK
• Comparable PNF/PDF for livers and long-term liver
  function tests
• Too soon for any conclusion regarding long-term
  biliary complications, but suggestion of less
  severe changes
• Similar early outcomes for kidneys and pancreases